大鲵蛙病毒US22家族基因的TaqMan MGB荧光定量PCR检测方法的建立及应用

为了进行大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)的早期诊断,以US22家族基因作为靶基因,建立大鲵蛙病毒Taq Man MGB实时荧光定量PCR检测方法,同时对该方法进行特异性、敏感性、重复性评估,并对73份未知大鲵血液样品进行检测。结果显示,标准曲线在所选浓度范围内具有良好的线性关系,相关系数为0.997;该方法对大鲵蛙病毒的检测有高度特异性,与其他8种病毒或细菌之间均无交叉反应;灵敏性良好,能检测到2.00×101copies/L的标准品浓度,比普通PCR高1 000倍;批内和批间重复的变异系数均小于2%,重复性好;在73份未知样品检测中,Taq Man MGB实时荧光定量PCR能够检测到更低浓度的病毒,较常规PCR的敏感性更佳。鉴于该方法快速、特异、敏感、可重复、可定量的优点,对大鲵蛙病毒早期潜伏感染的快速诊断具有重要意义。     

兔出血症病毒Yaan株衣壳蛋白VP60基因的克隆和序列分析

采用RT-PCR方法扩增兔出血症病毒(RHDV)Yaan株衣壳蛋白VP60基因,将扩增片段克隆到pMD 18-T载体上,经酶切及PCR鉴定后测序。结果显示,VP60基因全长1 740 bp,编码579个氨基酸。将Yaan株与其他RHDV分离株进行比较,结果显示,核苷酸同源性为90.5%~97.5%,氨基酸同源性为94.3%~99.5%,氨基酸变异多发生在衣壳蛋白C、E区,表明毒株具有高度保守性;同时对Yaan株与国外标准株AST/89的VP60蛋白氨基酸变异及其亲水性、柔性区、抗原区和表面结构进行了比较分析。     

赤羽病病毒N基因硫氧还蛋白融合表达载体的构建与表达

为获得赤羽病病毒 (Akabanevirus ,AKAV )的核蛋白重组抗原 ,根据其基因序列(SmRNA)设计合成了 1对特异性引物 ,对AKAVN基因进行RT PCR扩增 ,产物大小约为 6 96bp ,回收纯化后 ,将其克隆至 pMD18 T质粒载体中 ,经核苷酸序列分析 ,与已报道的多株AKAV编码核衣壳蛋白N的SmRNA基因比较后发现 ,所测定毒株的核苷酸序列与TIN毒株的同源性高达 98.3% ,推测的氨基酸同源性为 97.6 % ,证实为AKAV的N基因。将该基因片段亚克隆插入pBAD/ThioTOPO表达载体 ,转化TOP10细胞 ,成功地构建了AKAVN基因重组表达载体。经L araboinose诱导表达 ,可稳定、高效地表达AKAV核蛋白抗原。表达蛋白为融合蛋白 ,分子质量约 4 2 .5ku ,其表达产量约占菌体总蛋白的 2 8% ,融合蛋白中含有AKAV特异性核蛋白抗原 ,可作为ELISA包被抗原     

犬细小病毒JS12株VP2基因的分子特征及其在大肠杆菌中的表达

从犬细小病毒病免疫预防失败病犬体内分离到1株犬细小病毒(CPV),命名为CPV-JS12株。病毒可凝集猪的红细胞,对乙醚不敏感,pH3不能灭活病毒,pH10可使病毒失去感染性,56℃作用60min病毒仍有活性,80℃作用30min可灭活病毒。序列分析时CPV-JS12为CPV-2a亚型,VP2基因全长1755nt,编码584aa,与国外CPV毒株VP2基因的核苷酸序列和氨基酸序列的同源性分别在98.9%~99.6%和98.3%~99.7%之间,与国内和周边地区毒株VP2基因的核苷酸序列和氨基酸序列的同源性分别在99.0%~99.7%和97.9%~99.7%之间;16个中国分离株处在不同组,CPV-JS12株与韩国毒株DH426处在同一分支上,亲缘关系较近。对CPV-JS12与免疫毒株VP2蛋白的立体结构分析比较,发现有9处氨基酸残基变异,其中6处位于衣壳蛋白表面,5处位于抗原表位区。将VP2基因在大肠杆菌中表达,重组VP2蛋白的分子质量为67.0ku,主要以包涵体的形式存在;Western-blot分析中重组VP2蛋白可与CPV阳性血清发生特异性反应。以纯化的重组VP2蛋白为抗原建立的CPV抗体间接ELISA检测方法具有良好的特异性。     

犬瘟热病毒RT-nested PCR检测方法的建立与应用

根据犬瘟热病毒(CDV)弱毒株Onderstepoort的核衣壳蛋白(NP)基因序列设计了套式引物,建立了RT-nested PCR检测方法。特异性试验表明,该法可以特异扩增出CDV NP基因片段,但从RNA病毒狂犬病病毒(RV)、犬副流感病毒(CPIV)、犬冠状病毒(CCV),DNA病毒犬细小病毒(CPV)、犬腺病毒(CAV)及正常Vero细胞中均不能扩增出条带。敏感性试验表明,RT-PCR可以扩增10-6稀释度的病毒RNA,而建立的RT-nested PCR可以扩增到10-9稀释度的病毒RNA,后者的敏感性明显高于前者。用RT-nested PCR法检测了2006年我国东北地区临床表现犬瘟热症状的病例19例,检出率达90%,明显高于RT-PCR的检出率(45%)。部分病例扩增片段的测序结果表明,CDV NP基因出现个别碱基变异。研究结果表明,建立的犬瘟热病毒RT-nested PCR方法敏感、特异,适用于动物犬瘟热的快速诊断。     

H5N1亚型高致病性禽流感病毒NS基因的克隆及序列分析

采用RT PCR技术对4株H5N1亚型高致病性禽流感病毒的NS基因进行了扩增,将获得的PCR产物分别与T载体连接,获得了4个毒株的NS基因阳性克隆。序列分析结果显示,分离毒株间的核苷酸同源率为90.2%～98.7%,氨基酸同源率为82.6%～97.8%。与其他毒株比较A Goose HLJ P46 2003(H5N1)和A Goose HLJ G2 2003(H5N1)株NS基因在第264～278位处发生了15个核苷酸缺失;A Goose Jilin W2 2004(H5N1)株NS1基因蛋白编码区的第652位碱基发生T C突变,成为终止密码子,造成NS1蛋白的C末端有13个氨基酸缺失。证实不同基因型的H5N1亚型禽流感病毒在我国家禽中同时存在;H9与H5亚型流感病毒基因间存在着广泛的遗传交换。     

应用PCR检测山羊痘病毒

初步建立了用于诊断山羊皮肤组织中山羊痘病毒的PCR方法。试验设计了 2对引物 ,用以分别检测山羊痘病毒的特异性基因和α 微管蛋白基因。克隆到的DNA扩增产物的序列与GenBank中收录的序列同源性达到 98%。试验表明 ,用PCR方法可快速检测样品中的山羊痘病毒。     

犬瘟热病毒TN野毒株核蛋白基因的克隆与序列分析

根据GenBank中收录的犬瘟热病毒(CDV)Onderstepoort株核蛋白基因(N)序列,设计了1对特异性引物,用该引物对分离的TN野毒株进行了RT PCR扩增;将扩增得到的PCR片段纯化后与pGEM T质粒连接,得到重组质粒pTN。经核苷酸序列测定,CDVTN株N基因的ORF全长1569bp,编码523个氨基酸;将TN野毒株与GenBank中收录的其他CDV毒株进行比较,N基因核苷酸序列的同源性为94%～99%,推导的N蛋白氨基酸序列的同源性为97%～99%。TN株与Onderstepoort疫苗株氨基酸序列的差异主要集中在N端(17～159位)和C端(408～519位),中间的区域则高度保守。此外,在CDVN蛋白N端281～289位上也发现存在与麻疹病毒(MV)N蛋白完全相同的Y P A L G L H E F9肽序列,推测可能是诱导CTL活性的靶蛋白的抗原表位之一。氨基酸组成分析发现,推导的N蛋白氨基酸序列中亮氨酸、丝氨酸和异亮氨酸所占比例很高,提示该蛋白具有高度螺旋和缠绕的分子结构。     

犬瘟热病毒TaqMan实时荧光定量RT-PCR检测方法的建立及应用

为建立快速检测犬瘟热病毒的诊断方法,根据GenBank中犬瘟热病毒核衣壳蛋白(NP)基因的保守序列设计1对特异性引物和1条特异性探针,通过对反应体系和反应条件的优化,建立了TaqMan实时荧光定量RT-PCR检测方法。结果显示,该方法对犬瘟热病毒最低检出量为80copies/μL,是常规RT-PCR的100倍;与其他犬类病毒不发生交叉反应;组内、组间变异系数均小于5%。对收集的67份临床样品进行检测,阳性检出率为64.2%,而常规RT-PCR的阳性检出率为44.8%。研究结果表明,建立的TaqMan实时荧光定量RT-PCR具有良好的敏感性、特异性和稳定性,为犬瘟热的早期诊断提供了技术手段。     

番鸭呼肠孤病毒ZJ99株σC蛋白基因的克隆与序列分析

参考已发表的番鸭呼肠孤病毒(mDRV)的S4序列设计了1对扩增σC蛋白基因的引物,对mDRVZJ99株的RNA抽提物进行RTPCR扩增,获得了特异性的扩增片段。将PCR产物克隆测序,序列经同源性比较,发现mDRVZJ99株σC蛋白基因序列与福建MW9710株对应基因的同源性为99.5%,与法国89026株对应基因的同源性为94.2%,与法国89330株对应基因的同源性为95.0%;相应的氨基酸序列同源性分别为98.9%、94.1%、93.7%。基于σC基因及其推导氨基酸序列的蛋白质结构及物理化学性质预测结果,与国外学者对mDRVσC蛋白的报道相似。     

墨西哥应对贸易争端的经验与启示

墨西哥是开放程度和保护程度均较高的发展中大国,在处理国际贸易争端和保护自身利益的进程中,积累了丰富的经验,建立了较完善的组织体系和法律体系。中国是遭受墨西哥反倾销最多、受伤害程度最深的发展中国家。墨西哥对中国商品的反倾销制裁至少产生了两方面的影响一是中国向墨西哥出口的直接贸易损失;二是扩散效应,其他拉美国家参照墨西哥的做法,对中国商品进行反倾销制裁。我们应重视和研究墨西哥的经验,认真研究墨西哥的法律框架,建立产业对话机制,探索建立解决贸易争端机制的可能性。     

Handle with care: engaging with faith-based organisations in development

Religious faith has always had an intense but uneasy relationship with development. Donors are currently seeking greater engagement with faith-based organisations (FBOs). This positive shift needs careful consideration. Faith can be a powerful – but flammable – fuel for change. FBOs are highly diverse and complex. Donors therefore need to handle them with understanding and care. This article outlines both the major concerns about faith in development and also the potential ‘value-added’ of FBOs. It charts growing interest yet residual ambivalence on the part of donors towards faith in development. It presents the practical challenges and suggests ways forward for both donors and FBOs.     

论当前广西与泰国的经贸关系——兼谈与云南和泰国经贸关系的比较分析

在发展同泰国的经贸关系中,广西与云南具有许多相似点,例如广西与云南的区位优势都很明显,都是中国通向东南亚的桥头堡,与泰国距离都很近;根据专家的研究,云南的傣族、广西的壮族与泰国的民族都是"同源异流",双方习俗相似、语音相近,两省区都有不少华侨华人在泰国.更重要的是,云南与广西在产业结构上都是工业不发达以农业为基础的省份,两省区与泰国产业结构趋同和产业水平相近,产业内贸易在双方贸易总额中所占比重很大.在这样相同的背景下,对云南与泰国的经贸关系和广西与泰国的经贸关系进行比较分析,找出其中的差距,借鉴云南的经验,挖掘自身的优势,对于进一步推动广西与泰国的经贸关系的发展无疑具有重要的意义.     

Defending democracy with international law: preventing coup attempts with democracy clauses

In recent decades many regional inter-governmental organizations have adopted agreements committing all member states to maintain democratic governments, and specifying punishments to be levied against member states that revert to authoritarianism. These treaties have a surprisingly high enforcement rate – nearly all states subject to them that have experienced governmental succession by coup have been suspended by the relevant IGO(s). However, relatively little is known about whether these treaties are deterring coups. This article offers an original theory of how these international agreements could deter coups d’état, focusing on the way that a predictably adverse international reaction complicates the incentives of potential coup participants. An analysis of the likelihood of coups for the period of 1991–2008 shows that states subject to democracy were on average less likely to experience coups, but that this finding was not statistically significant in most models. However, when restricting the analysis to democracies, middle-income states with democracy clauses were significantly less likely to experience coup attempts. Moreover, the African democracy regime appears to be particularly effective, significantly reducing the likelihood of coup attempts for middle-income states regardless of regime type.     

Clemenceau's Contacts with England

Contacts between Clemenceau and England and the English between 1865 and 1920 are discussed, with special reference to a recent article by R. F. Hanks. The conclusion offered here is that Clemenceau developed his understanding of British political life principally through his friendship with Admiral Maxse and members of his family, who provided him with an entree into English political society. Aspects of the relationship between Clemenceau and the Maxse family can shed light on Anglo-French politics and Clemenceau's role.     

Negotiating with Your Nemesis

The assumption that negotiators can and should eradicate emotions from negotiating is unrealistic. Instead, effective negotiators know how to handle emotional outbursts including how to respond when the other negotiator evokes their nemesis. A nemesis is the hidden part of ourselves that we project onto others who push our hot buttons. When emotions are intense, understanding the possibility that you may be giving or receiving a projection can help you sidestep escalatory behavior. This article explores the concept of the nemesis and offers practical steps for confronting it as well as responding to others' emotions at the negotiating table.     

俄罗斯与巴西关系的形成和发展

俄罗斯和巴西是位于东半球和西半球上的两个大国。虽有大洋相隔 ,遥距万里 ,但两国具有悠久的历史联系。从第一个俄罗斯人跨入巴西国土至今已有 2 0 0多年 ,从两国正式建立外交关系至今已有 1 70多年。长期以来 ,随着外交关系的建立 ,两国在政治、经贸和文化方面的联系大大加强。尤其自 2 0世纪 90年代后半期至今 ,两国在外交、政治、经贸、科技和文化方面的联系更为密切 ,两国关系由一般伙伴关系升级为“长期战略伙伴关系”。一　两国关系的建立两国关系的渊源可追溯到 2 0 0多年以前。从1 8世纪初开始 ,在彼得大帝时期 ,俄罗斯就出现了介…     

Working with street children

In September 1990 we became involved with the Centre for the Defence of the Child (CDM) in Brazil, hoping to participate in a survey of the lives of street children that was being conducted by the group. The CDM, a branch of its parent organisation The Young Street Vendors' Association, takes on individual cases of street children, providing crisis management with social, psychological, and legal support. It was decided to start a project of regularly taking a team of staff on to the streets and into the areas where the street children were, with the aim of providing a programme of support and self-esteem building, principally for the street girls. This project proved a success with the street children and remains so following our departure from Brazil.